This invention relates to a gas cushion proportioning microsystem to proportion extremely small liquid volumes in the microliter and sub-microliter ranges.
In the known proportioning systems, a rough distinction is made between pipettes, dispensers, and multi-functional proportioners. Pipettes will redischarge the liquid volume received in one step. In dispensers, the discharge of the liquid volume received is performed stepwise. Multi-functional proportioners allow for both modes of operation. All of the system types also exist in a multi-channel design and permit to simultaneously perform several identical proportioning operations.
Conventional reciprocating-piston pipettes are designed as fixed-volume or adjustable-volume pipettes and operate in a volume range from somewhat less than 1 xcexcl up to about 10 ml. A piston is displaced in a cylinder, which moves a gas cushion in order to draw a liquid specimen into a pipette tip or to eject it therefrom. This has the advantage that the liquid does not come into contact with the reciprocating-piston pipette, but only contaminates the pipette tip. Therefore, this one mostly is designed as an expendable article (a xe2x80x9cdisposablexe2x80x9d), particularly in plastic, and will be exchanged against a clean pipette tip after use.
Proportioning by means of reciprocating-piston pipettes is burdened with various system-related errors. In adjustable-volume reciprocating-piston pipettes, a pipetting error arises by the fact that the weight of the liquid column xe2x80x9cis suspendedxe2x80x9d from the gas cushion and stretches it differently depending on the liquid volume adjusted. It is particularly for minor volumes being proportioned that proportioning accuracy is significantly affected because the pipette tip is wetted by the liquid being proportioned. Moreover, reciprocating-piston pipettes are incapable or only of a restricted capability of using open jets for small volumes being proportioned. No complete liquid discharge in an open-jet is reached already if the volumes being proportioned are of a few microliters and less. In addition, it is just for small volumes being proportioned that the reception of liquid considerably depends on the depth of immersion of the pipette tip into the liquid and the alignment of the reciprocating-piston pipette. This can be a significant source of errors especially in hand-operated pipettes. As a result, conventional reciprocating-piston pipettes are able to pipette small volumes only to a limited extent and at a relatively high imprecision and incorrectness.
Conventionally designed proportioning systems operating according to the direct-displacement principle include tips referred to as xe2x80x9csyringesxe2x80x9d with an integrated piston which is coupled to a driving device of the proportioning system. They are employed in a volume range from about 1 xcexcl to 50 xcexcl. Since there is no gas cushion and the piston is in a direct contact with the specimen being pipetted direct displacers are employed particularly if liquid of high vapour pressures, high viscosities or high densities is proportioned. This system type avoids the error resulting from the varying stretch of the gas cushion. However, proportioning small volumes at the accuracy required will be possible at best down to about 1 xcexcl. Also, direct-displacement syringes which can proportion such liquid volumes in an open jet are relatively expensive.
From WO 99/10099, a proportioning microsystem is known which comprises a reservoir, a membrane micropump the inlet of which is joined to the reservoir, an open-jet proportioner the inlet of which is joined to the exit of the membrane micropump, a proportioning port joined to the exit of the open-jet proportioner, and a proportioning control disposed in an operative communication with the membrane micropump and the open-jet proportioner. The membrane micropump is capable of pumping liquid into the open-jet proportioner from the reservoir. The open-jet proportioner is capable of dispensing the pumped-in liquid in an open jet. The open-jet capability makes it possible to proportion volumes being proportioned without no carry-over in the range of 1 nl up to a few microliters at high proportioning accuracies. If proportioning is to be made with another liquid this direct-displacement proportioning microsystem either needs to be cleaned to avoid carry-overs or to needs to be exchanged against a clean proportioning microsystem.
The same document has made known a microproportioning apparatus which has a membrane micropump which displaces a column of auxiliary liquid which, by means of a pipette piston, draws liquid into a pipette tip through a proportioning port or ejects it therefrom. The desired volume being proportioned is reached via the control of the known stroke volume of the membrane micropump. After a proportioning operation, the pipette tip and some of the column of auxiliary liquid may be discarded. In this system, the liquid flows off through the proportioning port and may be deposited on a substrate. It is impossible to deliver liquid in an open jet. Errors may occur particularly owing to wetting effects and, further, by differing depths of immersion and alignments. Therefore, its usability for proportioning extremely small liquid volumes is limited. In addition, there is a risk of contaminations because the pipette tip and the substrate contact each other.
Accordingly, it is the object of the invention to create a proportioning microsystem to proportion extremely small volumes in the microliter and sub-microliter ranges at an increased accuracy and a reduced risk of contamination and carry-over.
The object is attained by a gas cushion proportioning microsystem according to claim 1. Advantageous aspects of the system are indicated in the sub-claims.
The inventive gas cushion proportioning microsystem to proportion liquid volumes in the microliter and sub-microliter ranges comprises
1.1 a liquid reservoir including a storage space for the liquid being proportioned the boundary line of which is broken through by an outwardly leading liquid passage and a gas passage.
1.2 a gas displacement system which has a micropump to pump a gas, and a connection to the gas passage, and
1.3 a proportioning control disposed in an operative communication with the micropump to generate a negative pressure or positive pressure by actuating the micropump, and to apply the negative pressure or positive pressure to the liquid reservoir in order to receive liquid in the storage space through the liquid passage or to deliver it from said space.
A proportioning microsystem in the sense of the present application is a proportioning system which serves for proportioning small liquid volumes in the microliter range and sub-microliter range (from abt. 50 xcexcl to abt. 1 nanoliter). What is characteristic of such proportioning microsystem is micropumps which are designed in the microsystem technology. Their manufacture in the microsystem technology specifically comprises the use of the following materials: semiconductors and/or plastic and/or glass and/or ceramics and/or metals. Those are processed by means of appropriate manufacturing techniques of the microsystem technology or by microstructuring them, e.g. by lithography and etching processes (for semiconductors) or LIGA processes (for metals, plastics, and ceramics).
The micropump concerned may specifically be a membrane micropump. A membrane micropump in the sense of the present application is a pump with a cavity which is defined by at least one membrane with which an actuator (a drive) is associated. The actuator may specifically be a piezoelectric actuator. However, other actuators may be employed as well, e.g. thermal-action actuators. A membrane micropump may be with no valve so as to act as a displacement device to displace a volume during a stroke. However, it may be equipped with valves which are switched in such a way that several successive strokes of the membrane produce a volume flow. Passive-response valves, which are controlled by the pressures applied, generally allow of a unidirectional operation of the membrane micropump, i.e. a volume flow in one direction only. Active-response valves which are purposefully switched by a control device also allow of a bidirectional operation of the membrane micropump, i.e. a volume flow in different directions.
Other designs of the micropump may also be employed, particularly a gear micropump, an impeller micropump or a diffusion air micropump.
The invention is based on the displacement of a gas cushion (particularly an air cushion) by means of a negative-pressure or positive-pressure gas discharge. In the proportioning microsystem, to this end, a gas reservoir is xe2x80x9cproducedxe2x80x9d by a negative pressure or positive pressure, which may be some 100 mbar, by means of a gas-delivering micropump. The negative pressure or positive pressure may precisely be controlled via the pumping rate (i.e. the volume flow delivered) of the micropump or the volume displaced thereby. The dependence of the pumping rate or the volume displaced on micropump actuation is known or can be determined also for other designs of the micropump.
Generally, the negative pressure or positive pressure generated by the micropump may act directly on the liquid reservoir. The connection between the gas displacement system and the liquid volume may be designed with no valve means here. i.e. may be permanently continuous. Then, the negative pressure or positive pressure will be applied to the liquid reservoir by actuating the micropump. The micropump may be designed without valves or with valves here. A high positive pressure or negative pressure may be provided for specifically by an appropriately sized effective displacement area (e.g. a membrane area) of the micropump (on one or several membranes, for example). It is preferred that the connection has valve means. A particularly high negative pressure or positive pressure may be developed by blocking the valve means and actuating the micropump. If the valve means are opened the liquid reservoir may be acted on by the negative pressure or positive pressure, which causes liquid to be received in or to be delivered from the liquid reservoir. The valve means inside the connection or more valve means in an ventilation duct branching off therefrom may be active-response valves of the micropump at the same time.
Preferably, a gas reservoir of the gas displacement system is xe2x80x9cchargedxe2x80x9d by the micropump with a negative pressure or positive pressure. The gas reservoir may specifically be formed in the micropump and/or separately from it. Charging is performed prior to the reception or discharge of liquid. It is preferred that this is accomplished by several membrane strokes of a membrane micropump. Valve means disposed in the connection between the gas displacement system and the liquid reservoir separate the gas reservoir from the liquid reservoir and control pressure discharge into the liquid reservoir.
Unlike in conventional air cushion proportioning systems, proportioning is possible at a very high accuracy because the marginal conditions have been chosen to be particularly favourable and can be fixed precisely: The level of the negative pressure or positive pressure and its precise controllability, namely, make it possible to ensure that wetting or capillary effects the inherent pressures of which are small as compared to the negative pressure or positive pressure are overcome during the reception and discharge of liquid and, therefore, do not impair it. In addition, the positive pressure can be controlled so that liquid discharge may be effected in an open jet and exactly that liquid volume which need to be delivered will be delivered. Inherent variations in the gas filling temperature which are caused by gas discharge are uncritical with respect to the environment because of the differential pressure exclusively causing the reception and discharge. Moreover, this will avoid contaminations and carry-overs because the system operates with a gas cushion and discharge may be effected in an open jet without any contact with the substrate.
For accuracy in proportioning, it is advantageous that the system allows of a precisely timed control in applying the negative pressure or positive pressure to the liquid reservoir, which creates favourable conditions for liquid reception or liquid discharge. In a system using no valve means, this can be reached by controlling the micropump. In a system using valve means, the start of pressure application may be precisely controlled by opening the valve means. Terminating or relieving the negative pressure or positive pressure is possible particularly by closing the valve means because pressure compensation in the low-volume gas cushion still remaining above the liquid volume may be effected very rapidly and may be taken into account in determining or controlling the volume being proportioned. Preferably, the negative pressure or positive pressure may be relieved by opening a valve means in a ventilation connection of the gas displacement system to the environment. Further, it is possible to relieve the negative pressure or positive pressure acting on the liquid volume by means of an oppositely acting negative pressure or positive pressure which may be applied, in particular, by pumps or by connecting an appropriately xe2x80x9cchargedxe2x80x9d gas reservoir.
It is preferred that the liquid reservoir is abruptly applied to by the negative pressure or positive pressure or that the negative pressure or positive pressure is abruptly relieved, which makes it possible to attain that the advantageous pressure conditions prevail from the start to the end of the proportioning operation. The abrupt application or relief of the pressure conditions may be attained, in particular, by an abrupt membrane deformation or an abrupt opening or closure of valve means. Preferably, the negative or positive pressure gas discharge is performed in a pulselike manner and, therefore, is also termed xe2x80x9cpulse pressure methodxe2x80x9d. The pulse pressure method permits an increased accuracy in proportioning by employing an increased negative pressure or positive pressure the level which is precisely controllable, in a precisely determinable period of time.
After the abrupt relief of the negative pressure or positive pressure, the abrupt termination of the suction or discharge of liquid is caused by the forces which counteract the inertia forces of the liquid volume. This may be contributed to, in particular, by the frictional forces of the vessel walls of the liquid reservoir which act on the liquid volume as well as by interfacial forces between the boundary of the liquid reservoir and the liquid volume. This can be favoured, in particular, by the conformation, dimensions, and choice of materials for the liquid reservoir. However, it is also possible to cause the abrupt termination of the suction or discharge by applying an additional negative or positive pressure which counteracts the inertia forces and can be provided by means of a pump or gas reservoir. Thus, suction can be abruptly terminated by abruptly relieving the negative pressure and applying a positive pressure, and discharge can be terminated by abruptly relieving the positive pressure and applying a negative pressure. Once the liquid volume is stopped the additional positive or negative pressure may be relieved again. Thus, it is for the first time that it becomes possible to pipette, dispense, and handle extremely small liquid volumes in the microliter and sub-microliter ranges (particularly from 0.1 to 10 xcexcl) by means of a gas cushion system at a high accuracy (e.g. at a proportioning accuracy of 1%). Since the liquid exclusively comes into contact with the liquid reservoir this one may be advantageously designed as a relatively simple disposable which can be replaced with a clean liquid reservoir after use, which avoids carry-overs. By the way, discharging liquid in an open jet makes it possible to transfer liquid volumes with no contact between the system and the substrate, and avoids contaminations. A considerable reduction in reagent consumption is possible as compared to the initially mentioned classico-mechanical pipettes and dispensers and the initially mentioned proportioning microsystem. The invention permits applications especially in filling microtitration plates with extremely small reception volumes, feeding analytical microfluid systems (e.g. for capillary electrophoresis), applying reagents to biochips, etc.
The system is adapted to be used for both receiving and discharging liquid volumes. In an advantageous manner, however, it is adapted to be used exclusively for receiving or exclusively for discharging liquid volumes, e.g. if a determined liquid volume is destined for being processed in the system or gets into the system in another way. Hence, the invention also includes systems which enable both applications or only one thereof.
Each of the liquid volume which is received or discharged may be determined or controlled in different ways. Thus, it is possible to determine by tests under which system conditions (e.g. the level of the negative pressure or positive pressure, the points of time to switch the valve means, the operation or rest of the micropump, the pumping rate or volume displacement of the micropump) a certain volume of a certain liquid will be received or will be discharged by the system. The system may repeat the reception or discharge of this liquid volume at a high accuracy if it is operated under the same system conditions.
Thus, for example, the negative pressure in the gas displacement system and/or the period for which the valve means will open for the pipetting of a desired liquid volume may be controlled just so that the desired liquid volume is received. Further, the positive pressure and the opening period may be controlled just so that this certain liquid volume may be safely discharged in an open jet. At this point, the positive pressure may continue to be applied until the liquid volume is completely ejected. However, it is also possible to control the negative pressure in the gas reservoir and the opening period of the valve means in such a way that at least the liquid volume to be received will be received and, afterwards, to control the positive pressure and the opening period in such a way that exactly the preset liquid volume will be discharged. The latter approach may also be made in dispensing, in which process at least the sum of all liquid volumes to be discharged requires to be received initially. In addition, it may be possible to find the system conditions for the reception or discharge of several determined liquid volumes or determined volumes of several different liquids and to reference to them for the precise reception and discharge of appropriate liquid volumes.
According to an advantageous aspect, the system comprises a pressure sensor which preferably is compensated in temperature to detect the pressure in the gas displacement system with which the proportioning control is in an operative communication in order to determine the liquid volume received or discharged or to control it to a preset value. Thus, the sensor may determine a variation in the negative pressure or positive pressure after the valve means is opened, and may utilize the proportioning control, while making recourse to Boyle""s law or another gas condition equation, possibly in conjunction with equations describing the flow behaviour of the gas in the proportioning microsystem, for the calculation of the gas volume change which corresponds to the liquid volume received or discharged. Such determination of the liquid volume, however, may be influenced by fabrication-related variations and, hence, unknown variations of the gas volume in the system. According to the above aspect which is based on the determination by tests of the system conditions for the reception and discharge of determined liquid volumes, this aspect also can solely relate to the reception or discharge or to the reception and discharge of determined liquid volumes.
Particularly advantageous is an aspect according to which the proportioning control regulates the negative pressure or positive pressure in the gas displacement system in drawing in or discharging liquid by detecting the pressure prevailing therein by means of the pressure sensor and controlling the pumping rate or volume displacement of the micropump to a preset value and determines the liquid volume received or discharged via the pumping rate or volume displacement of the micropump which is known at this pressure. This aspect is based on the consideration that the gas volume delivered by the micropump for keeping constant the negative pressure or positive pressure in the gas displacement system corresponds very accurately to the liquid volume received. This makes it possible to advantageously utilize the regulation of the negative pressure or positive pressure to a preset value for the determination or control of each liquid volume which is received or discharged. At this point, the accuracy in proportioning is essentially determined by the accuracy in pressure detection by means of the pressure sensor. According to the above aspects, this aspect also can relate only to the reception or discharge or to the reception and discharge of determined liquid volumes.
The liquid volume to be received or discharged may be firmly predetermined or may be adjustable.
According to another advantageous aspect, the liquid reservoir has a liquid passage which is formed as a nozzle. The nozzle favours the attainment of a speed in the outlet which leads to the formation of an open jet. In addition, the cross-section in the storage space may be dimensioned to be so large that the flow of liquid essentially is of a stopple profile there and residual liquid will not be caused to remain by adhesion on the wall of the storage space. Further, intensified frictional forces may act on the liquid in the region of the nozzle which if the negative pressure or positive pressure is relieved will outweigh the inertia forces of the liquid and suppress any further reception or discharge of liquid. Further, the outlet port of the liquid reservoir, particularly on a nozzle, may be designed to be so small in cross-section that if the surface is hydrophobic the capillary forces will prevent the penetration of liquid into the outlet port up to a significant depth of immersion by a mere immersion thereof into the liquid. This makes it possible to achieve an independence of liquid reception on the depth of immersion that, specifically, is sufficient for portable units. In addition, a hydrophobic surface may avoid any adhesion that possibly exists in the nozzle while liquid is being discharged.
Apart from the micropump, one or several further components of the system may be designed in a microsystem technology, particularly the gas reservoir, the valve means, the proportioning control, the pressure sensor, the further valve means, the liquid reservoir, and all of the connections between the components or between these and the environment. This favours a miniaturization of the system and cost-efficient series production. Besides, the microsystem technological design may ensure that the components work at the desired speediness. In addition, extremely fine structures of a microsystem technological design may be manufactured at the accuracy which is required. It is particularly advantageous to arrange one or more components of the system on a microfluid board which may also comprise connections between the components or with the environment.
Miniaturizing the gas-carrying components of the system, e.g. by a microsystem technological design, is also advantageous because of the reasons which follow: While a negative pressure or positive pressure is applied to the liquid reservoir the negative pressure or positive pressure will abruptly drop because it has spread onto the liquid reservoir from the gas displacement system. This pressure drop should turn out to be as small as possible. In addition, it is also advantageous to design the gas reservoir with a small volume because it will then be easier for the pressure sensor to detect a change in the negative pressure or positive pressure due to liquid reception or discharge and, thus, the determination or control of the liquid volume received or discharged can be carried out more precisely. For these reasons, the dead volume, i.e. that air cushion volume which has spread over several components of the system, preferably will be dimensioned to be approximately equal to the maximum liquid volume to be received.